Here’s one to add to the “Who knew?” file, which is already a pretty large one in protein biology. There’s growing evidence that many proteins have a previously-unappreciated crosslinking chemistry available to them, a redox-sensitive N-O-S linkage that I think would have flunked me on a biochemistry test if I’d proposed it as an undergraduate (see diagram at right). These things bridge lysines and cysteines, and as you see they can be linear, cyclic, and a mixture of both.
These things were found by careful examination of protein X-ray data and distinguishing these bridges from the more expected hydrogen-bonding interactions. The initial evidence came from a handful of structures at extremely high resolutions (< 1Å), which is a level of detail you rarely get to investigate in proteins. Further complicated the situation is the way that the NOS bridge will be at various occupancy levels in a given crystal (that is, not every protein molecule in the crystal will have an intact bridge, which makes it harder to detect in the overall data). But working out the structural features of this linkage allowed for mining the Protein Data Bank to look for likely examples that hadn’t been detected before. Among the <2Å structures, several hundred examples were found where the N-S distance was too close for hydrogen bonding, but in just the right range for the N-O-S feature. And this result suggests that it’s probably a general feature that we’ve just been missing.
They’ve found in all sorts of places – some of them look like the artificial chemical “staples” that have been made inside single helices, while others bridge different helix motifs, crosslink a beta-pleated-sheet motif, or close a single-strand loop. These things can apparently form any time you have a Lys/Cys pair that can get close enough in the presence of either oxygen or some reactive oxygen species (and those two are in pretty good supply in the cell). It’s believed that the NOS structure dynamically modulates the function of both the Lys and Cys residues, and since these are hugely important side chains in protein function, we have apparently been blind to a whole layer of protein regulatory behavior. Catalytic residues in active enzyme sites, DNA-binding lysine residues, substrate recognition, regulation of Cys oxidation states – there are a lot of possibilities. One place to immediately start looking for NOS behavior would be in proteins that are known to be sensitive to oxidative stress, of course. And those are present all across living creatures, from bacteria that can switch between aerobic and anaerobic growth all the way up the multicellular organisms that have to deal with changes in oxygen levels under changing conditions of circulation or exercise.
So as this area continues to solidify, we’re clearly going to have a lot to think about in protein function (both in health and disease). New targets for drug discovery could open up. And the protein structure people will have some thinking to do, of course. I already wonder what the protein structure prediction programs have to say about these motifs – do they pick up on them as a possibility, or are they as blind to them as the rest of us have been? And taking a larger view, whenever something like this happens, I always stop to think about what other big, important, wide-ranging things about protein behavior and cell biology that we’re still missing. You know they’re out there. They always have been, and it’s going to be a very long time before we track them all down.
